Mycoplasma pneumoniae is a member of the class of cell wall-less prokaryotes Mollicutes, which includes the smallest and simplest free- living bacteria known to man. Mycoplasmas have limited biosynthetic capabilities, and many species have resorted to a parasitic lifestyle. As a consequence, mycoplasmas have a great impact on both public health and agriculture. M. pneumoniae is a pathogen of the human respiratory tract and the leading cause of pneumonia in older children and young adults. Adherence by M. pneumoniae to host cells (cytadherence) is pivotal to successful colonization and disease. Mycoplasma protein HMW1 is a phase-variable membrane protein that appears to have an accessory role in cytadherence. HMW1 exhibits an unusual subcellular distribution, being associated specifically with filamentous cell extensions and partitioning primarily, but not exclusively, with the cytoskeleton-like triton shell of M. pneumoniae. This proposal expands upon preliminary data which indicate that HMW1 is phosphorylated. The project addresses fundamental questions regarding the characterization of phosphorylation in vitro and in vivo, as well as the identification and localization of phosphorylated residues in HMW1. Standard techniques will be employed to assess incorporation of 32P into HMW1 during culture and in vitro. HMW1 will be analyzed biochemically to identify phosphorylated residues and localize those residues within the primary amino acid sequence. Finally, the phosphorylation of HMW1 will be examined for a correlation with its partitioning characteristics in Triton X-100 and with mycoplasma cytadherence. The model system offers a number of attractive features, including a target protein associated with an identifiable function (cytadherence), as well as the availability of variants lacking HMW1, of specific antibodies to HMW1, and of deduced amino acid sequence, that is currently being derived from the cloned gene for HMW1. The information gained from these studies will provide the necessary foundation for the long-term objective of defining the significance of HMW1 phosphorylation to protein function.